1. Field of the Invention
This invention relates to pluripotential embryonic stem cells and methods and compositions for making pluripotential embryonic stem cells.
2. Background Art
Primordial germ cells (PGCs) in the mouse are thought to be derived from a small population of embryonic ectoderm (epiblast) cells set aside at the egg cylinder stage prior to gastrulation (Lawson and Pederson, 1992), or even earlier (Soriano and Jaenisch, 1986). By 7 days post coitum (p.c.) about 100 alkaline phosphatase (AP) positive PGCs can be detected in the extra embryonic mesoderm just posterior to the definitive primitive streak (Ginsberg et al., 1990). These cells continue to proliferate and their number increases rapidly to around 25,000 at 13.5 days p.c. (Mintz and Russell, 1957; Tam and Snow, 1981). At the same time the PGCs migrate from the base of the allantois along the hind gut and reach the genital ridges by 11.5 days p.c. In the genital ridge, PGCs stop dividing at around 13.5 days p.c., and enter either mitotic arrest in the developing testis or meiosis in the ovary. In a few strains of mice, e.g. 129, this normal program can be disrupted if the male genital ridge from an 11.5 to 12.5 days p.c. embryo is grafted to an ectopic site such as the testis or kidney capsule. Under these conditions some PGCs give rise to teratomas and transplantable teratocarcinomas containing pluripotential embryonal carcinoma (EC) stem cells (Stevens and Makensen, 1961; Stevens, 1983; Noguchi and Stevens, 1982).
Previous studies have shown that steel factor (SF) and leukemia inhibitory factor (LIF) synergistically promote the survival and in some cases the proliferation of mouse PGCs in culture (Godin et al., 1991; Dolci et al., 1991; Matsui et al., 1991). However, under these conditions, PGCs have a finite proliferative capacity that correlates with their cessation of division in vivo. A similar finite proliferative capacity has been reported for oligodendrocyte-type 2 astrocyte (O-2A) progenitor cells in the rat optic nerve. In this case, PDGF is involved in the self renewal growth of O-2A cells (Noble et al., 1988; Raff et al., 1988). After a determined number of cell divisions, O-2A cells may lose their responsiveness to PDGF and start differentiating into oligodendrocytes. If both PDGF and basic fibroblast growth factor (bFGF) are added in culture, O-2A progenitor cells keep growing without differentiation (Bogler et al., 1990).
Since pluripotential embryonic stem cells (ES) can give rise to virtually any mature cell type they are of great value for uses such as creating genetically manipulated animals. However, according to the published scientific literature, it has previously been possible only to obtain ES cells from mice. These murine ES cells were obtained from cultures of early blastocysts. Attempts at isolating ES cells from other animals apparently have failed. One patent publication, Evans et al., published Apr. 5, 1990 under PCT Publication WO 90/03432, claims that pluripotential ES cells can be obtained from ungulate blastocysts in vitro. The application claims that these cells are expected to be epithelial and to have a very different morphology to mouse ES cells because ungulate embryos normally form an “embryonic disc”. This appears to be the basis of the allegation that the cells which they grow out of pig and cow blastocysts and which have a more epithelial morphology than mouse ES cells are, in fact, ES cells. However, mouse embryos also develop an epithelial layer of pluripotential embryonic ectoderm or epiblast cells. This layer is called an “egg-cylinder” rather than an “embryonic disc”. Therefore, there is apparently no strong embryological reason why the ungulate ES cells should have a different morphology to mouse ES cells. In addition, the evidence presented in the Evans appliction for the differentiation of the cow and pig putative ES cell lines into differentiated cell types in monolayer culture, in embryoid bodies and in tumors, is not convincing. Therefore, there is a great need to produce and maintain ES cells from a variety of different animals.
The present invention satisfies this need by demonstrating that, in the presence of bFGF, SF and LIF, PGCs continue to proliferate in culture and give rise to colonies of ES cells. These stem cells can give rise to a wide variety of mature, differentiated cell types both in vitro and when injected into nude mice and when combined with embryos to form a chimera.